Mutations in the glycosylation of proteins have dramatic effects on cell proliferation rate, cell sorting, pseudoplasmodial migration, spore coat formation, spore germination, and protein transport rate, in the cellular slime mold Dictyostelium discoideum. The long term goal of the project is to understand at the molecular level how glycosylation supports these cellular activities in normal cells. Analysis of a mutant that is conditional for GDP-Fucose synthesis has demonstrated that optimal cell proliferation rate and spore health depend on normal fucosylation. Examination of fucosylation pathways that may influence these processes has led to the partial characterization of a fucosylation reaction which occurs in the cytoplasm of Dictyostelium cells. Because of their compartmentalization, the acceptor fucoprotein (FP21), and the fucosyltransferase which modifies it (FP21-fucosyltransferase), are specially poised to influence these behaviors. Biochemical studies on fractionated cells have led to the conclusion that FP21 accumulates in the cytoplasm, that active FP21-fucosyltransferase accumulates in the cytoplasm, and that the fucosyltransferase has a very high affinity for the sugar nucleotide donor, GDP-Fucose. This has led to the development of a model that FP21 is synthesized and accumulates in the cytoplasm, and that it is glycosylated by resident glycosyltransferases, which are synthesized there, from cytoplasmic pools of sugar nucleotides. The specific hypotheses to be tested are that FP21 is synthesized in the cytoplasm, that it is fucosylated in the cytoplasm by an alpha(1-4)fucosyltransferase which is also synthesized there, that FP21 possesses unique structural features that mark it as a fucosyl acceptor for the FTase, and that this reaction is conserved in other organisms. These hypotheses will be tested by developing antibodies to study the compartments of biogenesis of FP21 and the fucosyltransferase, utilizing immunomicroscopy to confirm localization in intact cells, comparing the predicted amino acid sequences of the primary translation products with those of known cytoplasmic, non-membrane proteins, determining the structure of the FP21 oligosaccharide, and carrying out competition studies on the fucosyltransferase reaction. Once FP21 and its fucosyltransferase are defined in these ways, there will be a basis to directly test their role in proliferation and spore health, and to identify glycoproteins that are modified in this way in other organisms. It is important to understand cytoplasmic glycosylation because of its tremendous regulatory potential, as evidenced by the roles of phosphorylation of Ser and Thr residues for activity and conformation of modified proteins. A second reason for characterizing this pathway relates to the increasing attention given to the analysis of fucoconjugates and fucosylation activities in biopsies and biological fluids, as markers for cellular activity in pathology. A rational interpretation of these analyses would benefit from an understanding of the multiple potential cellular origins of these substances.